The present invention relates to a process for the isolation of 2-deoxy ecdysterone from Zoanthus sp useful as an oxytocic agent. The invention also relates to a method of inducing uterus contractions in mammals by administering an effective amount of 2-deoxy ecdysterone obtained from Zoanthus sp.
The animal Zoanthus sp is a marine invertebrate belonging to the Phylum Coelenterata, class Anthozoa and order Zoanthidea. Several bioactive compounds have been isolated from this source. Most important among them is the highly toxic palytoxin (LD50 in mice=0.15 mg/Kg, Moore R E and Scheuer P J, 1971, Science, 172:495), which is also an active cardiovascular agent. Other compounds reported from this group of animals are: homopalytoxin, bishomopalytoxin, neopalytoxin, deoxypalytoxin (Quinn R. J in Bioorganic Marine Chemistry, Vol 2, 1988, Edited by P J Scheuer, pp 25-27), two pyrazine derivatives: palythazine and isopalythazine, and a group of nitrogen pigments, paragracine I-VII (Bakus G J, Targett N M and Schulte B, 1986 ,J Chem Ecol, Vol 12:951-987), 3-norpseudozoanthoxanthin (Cimino G, De Stefano S, Fenical W, Lin G H Y, Wing R M, Radick P and Sima J J, J Am Chem Soc, 1973, 95:4049) besides the alkaloids, zoanthamine, zoanthenamine and zoanthamide (Rao C B, Anjaneyulu A S R, Sarma N S, Venkateswarlu Y, Rosser R M, Faulkner D J, 1985. J Org Chem, 50:3757-3760).
Ecdysones and ecdysterones are moulting hormones found in crustaceans and insects (D J Tighe-Ford, 1977. Hormonal aspects of Barnacle antifouling research. In Marine Natural Products Chemistry, D J Faulkner and W H Fenical (eds), Plenum Press, 383). The zoanthid Gerardia savaglia was found to contain large quantities of the crustacean molting hormone, ecdysterone (Sturaro A, Guerriero A, De Clauser R and Pietra F, Experientia, 1982, 38:1184-1185). The related compounds, Palythoalones A and B (ecdysteroids) from the marine zoanthid Palythoa australiae have recently been isolated. (Shigemori H; Sato Y; Kagata T; Kobayashi J, 1999. Palythoalones A and B, new ecdysteroids from the marine zoanthid Palythoa australiae. J. Nat. Prods, 62(2):373).
Agents that stimulate the pregnant uterus and are of importance in obstetrics are:
1-Oxytocics: Oxytocin and ergometrine
2-Prostaglandin E and F type compounds
Oxytocin causes regular coordinated uterine contractions each followed by relaxation. It is the drug of choice used to induce or augment labour when the uterine muscle is not functioning adequately (Rang H. P., Dale M. M; Ritter J. M., Pharmacology, third edition, pp 470, Churchill Livingstone, 1995). It is particularly used in those cases such as diabetes, isoimmunisation, hypertensive states, intrauterine growth retardation, placental insufficiency in which continuation of pregnancy is considered to be more harmful to the mother and/or foetus than the risks of delivery or pharmacological induction (Andrew J. Nichols; Robert R. Ruffolo, Jr., Uterine Pharmacology, in Principles of Pharmacology. Basic Concepts and Clinical Applications. Ed. Paul L. Munson. pp 202, 1995). Oxytocin may also be used in the treatment of postpartum hemorrhage resulting from uterine atony (Andrew J. Nichols; Robert R. Ruffolo, Jr., Uterine Pharmacology, in Principles of Pharmacology. Basic Concepts and Clinical Applications. Ed. Paul L. Munson. pp 202, 1995). Historically the ergot alkaloids were the first agents used to initiate or accelerate parturition. In modem obstetrics, oxytocin is used for this function and ergot alkaloids are most often used for treatment of postpartum hemorrhage (Cornelia R. Graves, Agents that cause contraction or relaxation of the uterus, in Goodman and Gilman""s, The Pharmacological basis of therapeutics. Eds. Perry B. Molinoff and Raymond W. Ruddon, pp. 939, 1996). Specific receptors for oxytocin in human myometrium have been identified and differences in receptor density at various stages of labour also have been noted (Bossmar T; Akerlund M; Fantoni G; Szamatowicz J; Melin P; and Maggi M., Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: effects of the oxytocin antagonist atosiban. Am. J. Obstet. Gynecol. 1994, 171: 1634-1642, 1994). Oxytocin has dual effects on the uterus. It regulates the contractile properties of myometrial cells and elicits prostaglandin production by endometrial/decidual cells.
Prostaglandins currently used in obstetrical practice include PGE2, PGF2xcex1 and the synthetic derivative 15-methyl PGF2xcex1. More recently, the PGE1 analog, misoprostol, has been under clinical investigation for use as an abortifacient and cervical ripening.
The major use of PGE2 (dinoprostone, Prostin E2) and 15-methyl PGF2xcex1 (Carboprost, Hemabate) currently approved in the U.S.A is for the performance of mid-trimester abortions. 15-Methyl PGF2xcex1 is also used as an alternative to ergonovine or oxytocin in the treatment of postpartum hemorrhage. In addition, numerous studies have supported the beneficial effect of locally applied PGE2 as a cervical ripening agent (Buchanam D; Macer J and Yonekura M. L., Cervical Ripening with Prostaglandin E2 Vaginal Suppositories. Obstet. Gynecol. 1984, 63:659-663).
Prostaglandins E2 and F2xcex1, presently used for clinical purposes are not very stable. For example, radiolabelled PGE2, when injected into human volunteers, about 50% of the radioactivity is excreted in the urine within 5 hours. The corresponding value for PGF2xcex1 is around 90% (W P Collins; E A Willman, 1978. Prostaglandins and uterine functions, In Topics in hormone chemistry, W R Butt (eds), Ellis Horwood Publishers, 183). At least 15 compounds have been identified in both the studies by mass spectrometry.
The main objective of the present investigation is to provide a process for the isolation of 2-deoxy ecdysterone from Zoanthus sp.
Another objective of the investigation is to find out the unknown property of the compound, 2-deoxy ecdysterone, isolated and purified from Zoanthus sp in inducing uterus contractions.
Still another objective of the invention relates to a method of inducing uterus contractions in mammals, said method comprising administering in known manner an effective amount of 2-deoxy ecdysterone which isolated and purified from Zoanthus sp.
Yet another objective of the invention is to provide a stable bioactive compound which may be useful in obstetrics, to induce or augment labour, to control postpartum uterine atony and hemorrhage, to cause uterine contraction after cesarean section or during uterine surgery or to induce therapeutic abortion.
The present investigation undertaken by the inventors has led to the development and standardization of a facile method for the isolation and purification of 2-deoxy ecdysterone from a new source, Zoanthus sp using flash chromatography over silica gel and gel permeation chromatography over Sephadex LH-20. The invention further provides a novel biomedical use of this compound as a potent oxytocic agent. The invention has established that this compound is more potent than the standard oxytocin and PGF2xcex1 at the concentration of 200 xcexcg/ml. The compound, 2-deoxy ecdysterone is also very stable and soluble in aqueous solvents and may have wider therapeutical applications.
The compound, 2-deoxy ecdysterone also known as 2-deoxy xcex2-ecdysone or 2-deoxy-20-hydroxy ecdysone, was found to be effective in inducing contractions in the guinea pig uterus. It was found that the percentage of active component oxytocin contained in 50 xcexcg/ml of compound amounts to 80.8%, that at 100 xcexcg/ml to 92.3% which further increases to 118.0% at concentration of 200 xcexcg/ml. Uterine contractions produced by the compound were also compared with the standard contraction produced by prostaglandin (PGF2xcex1). It was observed that for the active compound, the percentage of active component PGF2xcex1 contained in 50 xcexcg/ml of compound amounts to 69.4%, that at 100 xcexcg/ml to 82.3% which further increases to 114.1% at concentration of 200 xcexcg/ml. Possible use of the compound in obstetrics could be to induce or augment labour, to control post partum uterine atony and hemorrhage, to cause uterine contraction after cesarean section or during uterine surgery or to induce therapeutic abortion.